The SE33 locus is one of the most polymorphic markers used in human identification. However, it also possesses a higher mutation rate than other STR loci which results in multiple microvariants both within the repeat and in the flanking regions. Such flanking region mutations can generate discordant allele calls between multiplexes using different primer pairs. Classically such discordance is due to a mutation within a primer binding site. However, a discordance observed in samples of primarily West African descent between the PowerPlex® ESI 17 and ESX 17 Systems does not fall in this category. The root cause is a set of up to three separate SNPs residing within the PowerPlex® ESI 17 SE33 amplicon, which are capable of disrupting a stem-loop structure present in the wild-type sequence. These mutations retard the migration of the amplicon on capillary electrophoresis by 0.6 to 0.9 bases relative to the wildtype amplicon. As single-stranded DNA with a partial double-stranded region is known to migrate faster these data are consistent with a stem-loop structure forming in the wild-type sequence post-electrokinetic injection while the mutants are unable to form this structure and hence migrate normally. Formation of a stem-loop (even one with a ΔG of only –5.77kcal/mol) is due to an inability to precisely control the temperature on the exposed cathode end of an array, thereby resulting in an incomplete denaturing environment. To alleviate this discordance we developed a new SE33 primer set for inclusion in the PowerPlex® ESI 17 Pro System. This change allows robust amplification of DNA samples containing these SNPs without the migration shift caused by these mutations. As the 3′ end of these new primer pairs are of the exact same sequence as those found in the PowerPlex® ESX 17 System, there is expected to be minimal if any affect on concordance. Finally the charges to this primer pair have been shown to eliminate nonspecific amplification seen in some casework samples with the PowerPlex® ESI 17 System.